Mono (2-ammonium-2-hydroxymethyl-1,3-propanediol)(2r,cis)-1,2-ep-oxipropyl-phosphonate with improved characteristics of stability and processing

ABSTRACT

A process is described for the preparation of mono (2-ammonium-2-hydroxymethyl-1,3-propanediol) (2R,cis)-1,2-epoxypropyl-phosphonate with improved characteristics of stability, of processing for the preparation of pharmaceutical forms and of stability of the pharmaceutical forms that contain it.

The present invention concerns the compound mono(2-ammonium-2-hydroxymethyl-1,3-propanediol)(2R,cis)-1,2-epoxypropyl-phosphonatewith new physico-chemical characteristics, the process for itspreparation and pharmaceutical compositions that contain it as an activeingredient.

The above mentioned compound (hereinafter indicated as FT) is the monosalt of trihydroxymethyl-aminomethane (THAM) with(2R,cis)-1,2-epoxypropyl-phosphonic acid, a compound endowed antibioticactivity known as Fosfomycin (Merck Index, X Edition, page 607, no.4137).

The compound FT of formula ##STR1## was described for the first time inEuropean patent No. 0 027 597 (Zambon S.p.A.), was developed for themonodose treatment of urinary tract infections and is commercialized inItaly with the trade mark "Monuril" which distinguish a pharmaceuticalform in water soluble granulate.

The process for the preparation of FT described in the European patentNo. 0 027 597 consists in reactingbis-(2-ammonium-2-hydroxymethyl-1,3-propanediol)(2R-cis)-1,2-epoxypropyl-phosphonate (i.e. the bis-salt of THAM withFosfomycin) with p.toluenesulfonic acid in ethanol.

The bis-salt of THAM with Fosfomycin was described in British patent No.2 025 975 or in U.S. Pat. No. 4,727,065 both in the name of ZambonS.p.A.

The compound FT prepared according to European patent No. 0 027 597shows the following characteristics (see example 2):

melting point: 114°-116° C. (113-116)

pH: 3.77 (3.6-3.8)

surface area: 1.72 m² /g (1.0-2.9).

The above reported values are those of a sample selected as standard(Reference A) and the values between brackets show the minimum andmaximum variations of the parameters within 5 identical preparations.

Spanish patent No. 511,527 (Compania Espanola de la Penecilina yAntibioticos S.A.) specifically concerns a process of the preparation ofthe mono-salt of THAM with Fosfomycin (FT) as well as of the bis-salt ofTHAM with Fosfomycin.

Said process for the preparation of FT consists in reacting in analcoholic medium the mono-salt of (+)-alpha-phenethylamine of Fosfomycinwith THAM and sulphonic acid in the molar ratio 1:1:1. When the bis-saltof THAM with Fosfomycin is desired the three above said compounds areuse din the ratio 1:2:1. Compound FT prepared according to Spanishpatent No. 511,527 shows the following characteristics (see example 3):

melting point: 120°-122° C. (118-122)

pH: 3.8 (3.7-3.9)

surface area: 0.88 m² /g (0.85-1.25).

The above reported values are those of a sample selected as standard(Reference B) and the values between brackets indicate the minimun andmaximum variations of the parameters with 5 identical preparations.

U.S. Pat. Nos. 3,641,063 and No. 3,914,231 in the name of Merck describeFosfomycin and its salts as well as the purification of the productobtained by fermentation processes.

In example 7 of U.S. Pat. No. 3,641,063 and in example 11 of U.S. Pat.No. 3,914,231, which are identical to each other, a process for thepurification of the sodium salt of Fosfomycin carried out by threesubsequent chromatographic purifications is described.

In the first one, the sodium salt of Fosfomycin is eluted on anion-exchange resin by means of a buffer consisting of an aqueous THAMsolution.

We have verified that the elute from this chromatographic columncontains THAM and Fosfomycin in the weight ratio 350:1 and, by means ofmass-spectroscopy, we have verified that in the mixture no FT ispresent.

Accordingly, if it was desired to prepare FT before the presentinvention, it was possible to use the process described in Europeanpatent No. 0 027 597 which affords FT having the characteristic of theabove reported reference A or to use the process described in Spanishpatent No. 511,527 which affords FT having the characteristics of theabove reported Ref. B.

Fosfomycin exhibits relatively little stability mainly due to the easyopening of the epoxide, and it is sensitive to both humidity andtemperature.

This instability stands also for the mono-salt with THAM both preparedaccording to European patent No. 0 027 597 (Ref. A) and according toSpanish patent No. 511,527 (Ref. B) thus making necessary to useburdensome precautionary measures for the storage of the activeingredient (sealed containers with driers), for its processing intofinished pharmaceutical forms (operations carried out in controlledenvironments with R.H. lower than 25%) as well as for the stability ofthe finished pharmaceutical forms. This is particularly true for solidpharmaceutical forms such as "Monuril" the stability of which asgranulate is limited to two years.

We have now surprisingly found a new process for the preparation of FTwhich affords the product with physico-chemical characteristicsdifferent from those of FT obtained by previously known methods and withimproved characteristics of stability which allow for better storage ofthe active ingredient, an easier processing of the substance forpreparing pharmaceutical forms and an improved stability of thegranulate prepared from it.

The new process, which is one of the objects of the present invention,consists in preparing in a reactor at a temperature comprised between15° and 50° C. a methanolic solution ofbis-(2-ammonium-2-hydroxymethyl-1,3-propanediol)(2R,cis)-1,2-epoxypropylphosphonate(i.e. the bis-salt of THAM with Fosfomycin), an equimolecular amount ofmethanesulphonic acid, an equimolecular amount of mono(+)-alpha-phenethylammonium (2R,cis)-1,2-epoxypropyl-phosphonate (i.e.the mono salt of phenethylamine with Fosfomycin) and an amount of 2-3%in mols, with respect to the amount of THAM contained in the bis-salt ofTHAM with Fosfomycin of trihydroxymethyl-aminomethane (THAM), thesolution is then diluted with ethanol in amounts between 4:1 and 10:1with respect to the volume of the solution, and cooled at about 0° C.under a slow stirring characterized by the following parameters:

length of the anchor stirrer/diameter of the reactor higher than 0.7

specific power lower than 0.25 kw/m³.

The precipitate consisting of the desired product is collected. Thecrystallization is preferably seeded by adding a smaller amount of FT.

The yield is about 75% in pure FT.

The mother liquors can be reused as such or from them the bis-salt ofTHAM with Fosfomycin may be recovered by adding an equimolecular amountof THAM with respect to the non-precipitated FT. The thus obtainedbis-salt is reused. The above described process affords FT, which is anobject of the present invention, having the following characteristics:

melting point: 122°-124° C.

pH: comprised between 4.0 and 5.0

surface area: comprised between 0.2 and 0.5 m² /g.

Hereinafter we will indicate as FT* the product obtained by the abovedescribed process and having the above reported characteristics, inorder to distinguish it from the general abbreviation FT and from FTprepared by the process described in European patent No. 0 027 597 (Ref.A) or by that of the Spanish patent No. 511,527 (Ref. B).

The process for the preparation of FT* is reproducible and easilyindustrially carried out thus providing FT* in good yields and highpurity with the above reported chemical and physical characteristics arereproducible and remain always in the above values.

FT* with said characteristics is more stable than FT according to Ref. Aand Ref. B (see example 4) both with respect to humidity and temperatureand consequently can be stored in conditions not burdensome from anindustrial point of view.

Thanks to the characteristics of FT* it is possible to preparepharmaceutical forms in granulate under more simple and less burdensomeprocessing conditions (see example 5).

The pharmaceutical compositions thus obtained, which are another objectof the present invention, result to be more stable (see example 6) andallow to increase the expiry date of the pharmaceutical composition upto three years from its preparation, while a two years stabilitycharacterizes the granulate containing FT according to Ref. A presentlycommercialized.

Thus, the new above described process allows to obtain a product (FT*)having different chemical and physical characteristics endowed withimproved stability which allow a more simple and convenient storage ofthe active ingredient, with improved characteristics for its processingwhich allow a more simple and convenient preparation of solidpharmaceutical forms, in particular granulates, with improved stabilityalso in the form of pharmaceutical composition thus allowing to increasethe validity of the same.

It is clear to the person of ordinary skill in the art how thesecharacteristics have a relevant industrial value.

With the aim to better illustrate the present invention, we give thefollowing examples.

EXAMPLE 1 Preparation ofmono-(2-ammonium-2-hydroxymethyl-1,3-propanediol)(2R,cis)-1,2-epoxypropyl-phosphonate (FT*) according to the presentinvention.

A) To a mixture of the bis-salt of THAM with Fosfomycin (10.3 g; 27.07mmols), THAM (0.2 g; 1.65 mmols) and methanol (38 ml) methanesulphonicacid (2.6 g; 27.08 mmols) was added in 10 minutes. At the end of theaddition the temperature of the mixture was 42°-43° C.

The mono-phenethylamine salt of Fosfomycin (7.5 g; 27.07 mmols) was thenadded while keeping the temperature at 42°-43° C. After a completesolution, ethanol (162 ml) was added by keeping the temperature at42°-43° C. The mixture was then stirred according to the followingparameters:

length of the anchor stirrer:diameter of the reactor=0.8;

applied power 0.15 kw/m³

and a sample of FT (0.02 g) was seeded.

The mixture was slowly cooled at 0° C. in 3 hours and kept at thattemperature for 3 hours.

The precipitate was filtered and washed with ethanol (10 ml). The wetproduct was mashed for 15 minutes with ethanol (25 ml) in the samereactor used before with the same stirring speed used for thecrystallization.

The product was filtered, washed with ethanol (10 ml) and dried undervacuum at 50° C.

The desired product was thus obtained (10.5 g; 40.54 mmols, yield 75%,m.p.=122°-124° C.).

A sample of the product was subjected to the test for evaluating thesurface area by using a Area Meter apparatus of Stroohlein GmbH whichuses the process of absorption of nitrogen at low temperatures accordingto the B.E.T. method [J. Am. Chem. Soc., 60, 309, (1938].

The test was replied three times thus obtaining the following values ofsurface area 0.37; 0.36 and 0.36 m² /g.

A sample of the product was subjected to the test for the determinationof the pH by using a digital pH-meter Top Tronic equipped with glasselectrodes combined Metrohm code 6.0202.000. The apparatus wascalibrated at 20° C. with solutions of known pH (pH 7 and pH 3).

An aqueous 5% solution of FT* kept at 20° C. showed pH 4.5.

B) The reaction described under point A was repeated 9 times, also ondifferent scales, providing FT* having characteristics comprised in thefollowing values of the parameters:

melting point: 122°-124° C.

surface area comprised between 0. and 0.5 m² /g

pH comprised between 4.0 and 5.0.

EXAMPLE 2

The present example concerns the preparation of FT (Ref. A) according tothe procedure described in European Patent no. 0 27 597.

A) A solution of para-toluenesulphonic acid monohydrate (52.5 g; 275.19mmols) in ethanol (260 ml) heated at 75° C. was added under stirring toa mixture of the bis salt of THAM with Fosfomycin (100 g; 262.88 mmols)and ethanol (660 ml) kept at 75° C.

By first a solution was obtained, then a crystalline solid begun toprecipitate, and it was cooled under stirring in two hours at +3° C.

The precipitate was filtered under vacuum, washed with absolute ethanol(140 ml) at +10° C. and dried in oven under vacuum at 40° C.

FT was thus obtained (Ref. A) (53.7 g; 207.3 mmols, yield 78.9%, m.p.114°-116° C.).

A sample was subjected to the tests for the evaluation of the surfacearea and pH according to the methods described in Example 1, with thefollowing results:

surface area: 1.72 m² /g (average of 3 tests), pH=3.77.

B) The reaction described under point A was repeated 4 times thusobtaining FT having characteristics comprised in the following values ofthe parameters:

melting point =113°-116° C.

surface area comprised between 1.0 and 2.9 m² /g

pH comprised between 3.6 and 3.8.

EXAMPLE 3

The present example concerns the preparation of FT (Ref. B) according tothe procedure described in Example 2 the Spanish patent no. 511,527.

A) After complete dissolution of a mixture of para-toluenesulphonic acidmonohydrate (38.4 g; 201.87 mmols) methanol (400 ml) and THAM (23.8 g;196,69 mmols), the mono-salts of phenethylamine with Fosfomycin (55.4 g;200 mmols) was added.

The mixture was stirred at 30° C. up to complete dissolution, thenisopropanol (1000 ml) was added and it was left under stirring for 1.5hours at 20° C. The mixture was filtered and the precipitate was washedwith a 1:1 mixture of methanol and isopropanol (200 ml).

The precipitate was dissolved in methanol (200 ml) at 55° C. Isopropanol(200 ml) was added under stirring and the mixture was cooled at roomtemperature in 1 hour and filtered. The insoluble was washed with a 1:1mixture of methanol and isopropanol (100 ml).

The product was dried in oven at 50° C. under vacuum for 8 hours. FT(Ref. B) was thus obtained (39.1 g; 149.1 mmols, yield 74.6%, m.p.120°-122° C.).

A sample of the product was subjected to the analysis for evaluating thesurface area and the pH according to the methods described in Example 1,with the following results: surface area 0.88 m² /g (average of 3tests), pH=3.8.

B) The reaction described under point A was repeated 4 times thusobtained FT with characteristics comprised between the following valuesof the parameters:

melting point =118°-122° C.

surface area comprised between 0.85 and 1.25 m² /g

pH comprised between 3.7 and 3.9.

EXAMPLE 4 Determination of the stability of FT rough material,comparison between FT*, Ref. A and Ref. B.

General procedure

About 11 g of each product was distributed into a crystallizer (glasscrystallization vessel) having diameter of 13.5 cm so as to overlay itsbottom with a ca. 0.5 cm layer.

The crystallizers were contempouraneously stored into a thermoclymaticroom regulated at 25° C. and relative humidity (R.H.)=50±5% or in a roomregulated at 25° C. and R.H.=70±5%.

Samples were withdrawn at the beginning of the test and after apre-established time interval (24 hours for the samples stored atR.H.=50±5% and 6 hours for those stored at R.H.=70±5%).

The samples were analyzed to determine the titre in Fosfomycin accordingto the following procedures.

HPLC analysis according to the following chromatographic conditions:

apparatus Liquid Chromatograph Mod. 1081B equipped with refraction indexdetector and integrator Mod. 3880 both Hewlett Packard brand

column Nucleosil 10SB, 10 μm, 25 cm×4.6 mm i.d. Alltech brand

temperature of column 35° C. and of the detector 30° C.

eluent: 0.3M solution of KH₂ PO₄

flow: 1 ml/min.

A standard FT solution was prepared by dissolving 500 mg of product inthe eluent brought at 20 ml volume.

The product to be analyzed was dissolved in the eluent at 25 mg/mlconcentration.

20 μl of standard solution and of solution to be analyzed were injected,the injections were repeated and the areas were averaged. In the abovedescribed conditions the retention time of Fosfomycin is ca. 6 minutes.

The percentage of FT in the sample (Ts) was determined by the followingcomputation ##EQU1## wherein As=area corresponding to the analyzedsolution

Ar=area corresponding to the reference standard solution

Pr=weight (in mg) of reference FT

Ps=weight (in mg) of the sample.

T(%)=percentage titre of FT in the reference standard.

The method affords a linear response inside the concentration interval10-50 mg/ml.

The precision of the method for c=25 mg/ml is expressed by a variationcoefficient (VC)=±0.94%.

The results are reported in the following table.

                  TABLE 1                                                         ______________________________________                                        titre in Fosfomycin of the sample (initial titre = 100%)                      Conditions        Ref. A  Ref. B    FT*                                       ______________________________________                                        24 h, 25° C., 50 ± 5% RH                                                              93.93%  96.84%     98.84%                                    6 h, 25° C., 70 ± 5% RH                                                              94.6%   98.82%    100%                                      ______________________________________                                    

Drying rate of a granulate containing FT, comparison between FT*, Ref. Aand Ref. B.

The industrial production of pharmaceutical compositions in granulatecomprises a phase in which the active ingredient is granulated in thepresence of water and a phase in which the granulate is dried underheating in order to reduce its content of water. In the case of activeingredients relatively unstable with respect to the humidity andheating, the drying phase can be very critical if protracted in time.

It is thus important, for a good processing of a relatively unstableactive ingredient, that it could reach in a short time a sufficientdegree of dryness.

Samples of FT*, Ref. A and Ref. B were granulated with water in amountsof 12% by weight.

At the end of the granulation process each sample contained water inamounts of 7% by weight.

For the preparation of the finished pharmaceutical form and for itsstability it is necessary that the granulated FT contains water inamounts not higher than 0.3%.

The three granulated samples were thus dried at 50° C. and every 30minutes samples were withdrawn for evaluating their water contents.

After 60 minutes the granulate prepared from FT* reached the acceptablewater content (0.3%).

The granulates prepared from Ref. A and Ref. B, on the contrary, reachedthe water content of 0.3% after 210 minutes.

EXAMPLE 6 Stability of pharmaceutical forms in water soluble granulate,comparison between FT*, Ref. A and Ref. B.

Starting from the granulates obtained from FT*, Ref. A and Ref. B anddried up to a water content of 0.3% by weight, pharmaceuticalcompositions in hydrosoluble granulate were prepared according to thefollowing procedure.

The sieved granulate was additioned with saccharin, orange and mandarinflavour and with saccharose.

After admixture, the ingredients were distributed inpaper-polyethylene-aluminium-polyethylene bags, each one containing

    ______________________________________                                               FT      5.631 g                                                               Saccharin                                                                             0.016 g                                                               Flavour 0.140 g                                                               Saccharose                                                                            2.213 g                                                        ______________________________________                                    

For comparison purposes, bags prepared as above reported, starting fromFT*, Ref. A and Ref. B were stored in a conditioned environment andperiodically analyzed for ascertaining their stability. The analysis wasperformed by withdrawing the formulate from the bags, by grinding in amortar and by weighing the amount equivalent to about 500 mg of FT whichwas transferred into a 20 ml volumetric flask and brought to volume by0.3 m KH₂ PO₄.

The reference solution was prepared by exactly weighing about 500 mg ofreference FT and by dissolving in a 20 ml volumetric flask with theeluent solution.

An HPLC analysis was performed according to the procedure described inExample 4.

The precision of the method resulted to be 101.1% and is expressed by avariation coefficient (VC)=±0.70%.

The storage conditions and the results are reported in the followingtable 2.

                  TABLE 2                                                         ______________________________________                                        titre in Fosfomycin of water soluble granulates (initial                      titre = 100%)                                                                 Storage condltions Ref. A  Ref. B   FT*                                       ______________________________________                                         3 months, 40° C., 20% RH                                                                 93.9%   94.64%    97.27%                                   12 months, T and RH room values                                                                  96.18%  96.57%   99.6%                                     24 months, T and RH room values                                                                  --      95.42%   99.8%                                     ______________________________________                                    

What we claimed is:
 1. Themono-(2-ammonium-2-hydroxymethyl-1,3-propanediol)(2R,cis)-1,2-epoxypropyl-phosphonate having the following physico-chemicalcharacteristics:melting point: 122°-124° C. ph: comprised between 4.0and 5.0 surface area: comprised between 0.2 and 0.5 m² /g. 2.Mono-(2-ammonium-2-hydroxymethyl-1,3-propanediol)(2R,cis)-1,2-epoxypropyl-phosphonate prepared by steps consistingof:preparing in a reactor at a temperature between 15°-50° C., amethanolic solution ofbis-(2-ammonium-2-hydroxymethyl-1,3-propanediol)(2R,cis)-1,2-epoxypropyl-phosphonate,equimolecular amounts of methanesulphonic acid, equimolecular amounts ofmono (+)-alpha-phenethylammonium (2R,cis)-1,2-epoxypropyl-phosphonateand amounts of trihydroxymethyl-aminomethane (THAM) equal to 2-3% inmoles with respect to the amount of THAM contained in the bis-salt ofTHAM with Fosfomycin; diluting the solution with ethanol in amountscomprised of between 4:1 and 10:1 with respect to the volume of thesolution; and cooling the solution under slow stirring, wherein theratio of a length of an anchor stirrer:diameter of the reactor isgreater than 0.7 and the power is less than 0.25 kw/m³.
 3. A solidpharmaceutical composition containing as active ingredient themono-(2-ammonium-2-hydroxymethyl-1,3-propanediol)-(2R,cis)-1,2-epoxypropyl-phosphonatehaving the following characteristics:melting point: 122°-124° C. pH:comprised between 4.0 and 5.0 surface area: comprised between 0.2 and0.5 m² /g.
 4. A solid pharmaceutical composition according to claim 3 inthe form of water-soluble granulate.
 5. A solid pharmaceuticalcomposition containing themono-(2-ammonium-2-hydroxymethyl-1,3-propanediol)(2R,cis)-1,2-epoxypropyl-phosphonateprepared according to the process steps of claim
 2. 6. A solidpharmaceutical composition according to claim 5 in the form ofwater-soluble granulate.